Compounds, compositions and methods for stabilizing transthyretin and inhibiting transthyretin misfolding

ABSTRACT

Compounds, compositions and methods are provided for stabilizing transthyretin and for treating, preventing, or ameliorating one or more symptoms of transthyretin mediated diseases. In one embodiment, the compounds are biphenyls and related compounds.

RELATED APPLICATION

Priority is claimed herein under 35 U.S.C. 119(e) to U.S. provisionalpatent application Ser. No. 60/573,720, filed May 20, 2004, entitled“COMPOUNDS, COMPOSITIONS AND METHODS FOR STABILIZING TRANSTHYRETIN ANDINHIBITING TRANSTHYRETIN MISFOLDING.” The disclosure of theabove-referenced application is incorporated by reference herein in itsentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funds used to support some of the studies disclosed herein were providedby grant number NIH DK 46335 awarded by the National Institutes ofHealth. The Government may have certain rights in the invention.

FIELD

Provided herein are compounds, compositions and methods relatinggenerally to protein misfolding. More particularly, provided herein arebiphenyl compounds, compositions and methods for stabilizingtransthyretin, inhibiting transthyretin misfolding, inhibitingtransthyretin fibril and amyloid formation and treating amyloid diseasesassociated thereto.

BACKGROUND

Transthyretin (TTR) is a 55 kDa homotetrameric protein present in serumand cerebral spinal fluid. The function of TTR is to transportL-thyroxine (T₄) and holo-retinol binding protein (RBP). TTR is one ofgreater than 20 nonhomologous amyloidogenic proteins that can betransformed into fibrils and other aggregates leading to diseasepathology in humans. These diseases do not appear to be caused by lossof function due to protein aggregation. Instead, aggregation appears tocause neuronal/cellular dysfunction by a mechanism that is not yetclear.

Under denaturing conditions, rate limiting wild type TTR tetramerdissociation and rapid monomer misfolding enables misassembly intoamyloid, putatively causing senile systemic amyloidosis (SSA).Dissociation and misfolding of one of more than eighty TTR variantsresults in a wide variety of familial amyloidoses, including familialamyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC).

The TTR tetramer has two C₂ symmetric T₄-binding sites. Negativelycooperative binding of T₄ is known to stabilize the TTR tetramer andinhibit amyloid fibril formation. Unfortunately, less than 1% of TTR hasT₄ bound to it in the human serum, because thyroid-binding globulin(TBG) has an order of magnitude higher affinity for T₄ in comparison toTTR. Furthermore, the serum concentration of T₄ is relatively low (0.1μM) compared to that of TTR (3.6-7.2 μM).

SUMMARY

Provided herein are compounds that kinetically stabilize the nativestate of transthyretin, thereby inhibiting protein misfolding. Proteinmisfolding plays a role in a variety of disease processes, includingtransthyretin amyloid diseases. By inhibiting transthryetin misfolding,one can intervene in or treat such a disease, ameliorate symptoms,and/or in some cases prevent or cure the disease.

The compounds, compositions and methods described herein for treating,preventing, or ameliorating one or more symptoms of TTR amyloidosis. TTRamyloidosis typically leads to death in 5 to ten years, and untilrecently, was considered incurable. Liver transplantation is aneffective means of replacing the disease-associated allele by awild-type (WT) allele in familial amyloid polyneuropathy cases becausethe liver is typically the source of amyloidogenic TTR. While livertransplantation is effective as a form of gene therapy it is not withoutits problems. Transplantation is complicated by the need for invasivesurgery for both the recipient and the donor, long-termpost-transplantation immunosuppressive therapy, a shortage of donors,its high cost, and the large number of TTR amyloidosis patients that arenot good candidates because of their disease progression. Unfortunately,cardiac amyloidosis progresses in some familial patients even afterliver transplantation because WT TTR often continues to deposit. Nor iscentral nervous system (CNS) deposition of TTR relieved bytransplantation owing to its synthesis by the choroid plexus.Transplantation is not a viable option for the most prevalent TTRdisease, senile systemic amyloidosis (SSA), affecting approximately upto 25% of those over 80 due to the deposition of WT TTR and for familialcardiac amyloidosis, including carriers of V122I, a mutant identified in3.9% of African Americans.

In another embodiment, the compounds for use in the compositions andmethods provided herein have formulae I:

where Y is COOH, COOR⁵, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂, CONHSO₂Ar,CONHCH(R⁶)COOH, OH, CH₂OH or —CH₂)_(n)—C(R⁶)(NH₂)—COOH;

X¹ is O, S, S(O), S(O)₂, —O—N═CH— or NR¹¹;

R¹, R² and R³ are each independently selected from hydrogen, halo, OR⁵,OAr, OHet, OCH₂Ar, OCH₂Het, CN, B(OH)₂, COOH, CONR⁷R⁸, alkyl,—(CR⁹R¹⁰)_(n)OH, —(CR⁹R¹⁰)_(n)NR⁷R⁸, —(CR⁹R¹⁰)_(n)SH or CF₃;

Het is heteroaryl, optionally substituted with halo, OR, alkyl orhaloalkyl;

Ar is aryl, optionally substituted with halo, OR, alkyl or haloalkyl;

R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, arylor heteroaryl;

R⁴ is hydrogen, halo, OH, or alkyl;

R⁵ is alkyl, haloalkyl, cycloalkyl or heterocyclyl;

R⁶ is the side chain of a naturally occurring α-amino carboxylic acid;

R⁷ and R⁸ are each independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl;

R⁹ and R¹⁰ are each independently hydrogen, halo, alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

R¹¹ is hydrogen or alkyl; and

n is an integer from 0-3.

In one embodiment, Het is pyrimidine, pyridine or thiophene.

In one embodiment, the compounds have formulae I, with the proviso thatwhen Y is COOH and is in the 3 or 4 position, then R¹, R² and R³ are notCl, CF₃ or F. In another embodiment, the compounds have formulae I, withthe proviso that when Y is COOH, then R¹, R² and R³ are not allhydrogen. In another embodiment, the compounds have formulae I, with theproviso that when Y is COOH and is in the 2 position, then R¹, R² and R³are not Cl, CH₃ or CF₃. In another embodiment, the compounds haveformulae I, with the proviso that when Y is OH and is in the 3 or 4position, then R¹, R² and R³ are not all hydrogen. In anotherembodiment, the compounds have formulae I, with the proviso that when Yis OH and is in the 3 or 4 position, then R¹, R² and R³ are not fluoro.In another embodiment, the compounds have formulae I, with the provisothat when Y is CH₂OH, then R¹, R² and R³ are not all hydrogen. Inanother embodiment, the compounds have formulae I, with the proviso thatwhen Y is CH₂OH, then R¹, R² and R³ are not chloro.

Also provided herein are pharmaceutical compositions containing thecompounds provided herein.

Also provided are methods for the stabilization of transthyretin in atissue or in a biological fluid, and thereby inhibiting misfolding.Generally, the method involves administering to the tissue or biologicalfluid a stabilizing amount of a compound provided herein that binds totransthyretin and prevents dissociation of the transthyretin tetramer bykinetic stabilization of the native state of the transthyretin tetramer.

Thus, methods which stabilize transthyretin in a diseased tissueameliorate misfolding and lessen symptoms of an associated disease and,depending upon the disease, can contribute to cure of the disease. Alsocontemplated herein is inhibition of transthyretin misfolding in atissue and/or within a cell. The extent of misfolding, and therefore theextent of inhibition achieved by the present methods, can be evaluatedby a variety of methods, such as are described in the Examples and ininternational patent application publication no. WO2004/056315. Thedisclosure of the above-referenced application is incorporated herein byreference in its entirety.

Also provided herein is a method of treating, preventing, orameliorating one or more symptoms of a transthyretin amyloid disease,the method involving administering a therapeutically effective amount ofa compound provided herein. In one embodiment, the compound preventsdissociation of a transthyretin tetramer by kinetic stabilization of thenative state of the transthyretin tetramer. The transthyretin amyloiddisease can be, for example, familial amyloid polyneuropathy, familialamyloid cardiomyopathy, or senile systemic amyloidosis. Othertransthyretin amyloid diseases include but are not limited toAlzheimer's disease, spongiform encephalopathy (Creutzfeldt Jakobdisease), polyneuropathy, type II diabetes and medullary carcinoma ofthe thyroid (see, e.g., International Patent Application PublicationNos. WO 98/27972 and WO 95/12815).

Methods of treating, preventing, or ameliorating one or more symptoms ofa transthyretin mediated disease or disorder by administering a compoundprovided herein are provided. Transthyretin mediated diseases anddisorders include but are not limited to obesity (see, e.g.,International Patent Application Publication No. WO 02/059621).

Further provided is a method of stabilizing TTR tetramers using acompound or composition provided herein. Also provided is a method ofinhibiting formation of TTR amyloid using a compound or compositionprovided herein.

Also provided herein is use of any of the compounds or pharmaceuticalcompositions described herein for the treatment, prevention, oramelioration of one or more symptoms of a transthyretin amyloid disease(e.g., familial amyloid polyneuropathy, familial amyloid cardiomyopathy,or senile systemic amyloidosis).

Also provided herein is use of any of the compounds or pharmaceuticalcompositions described herein in the manufacture of a medicament for thetreatment, prevention, or amelioration of one or more symptoms of atransthyretin amyloid disease (e.g., familial amyloid polyneuropathy,familial amyloid cardiomyopathy, or senile systemic amyloidosis).

Articles of manufacture, containing packaging material, a compound orpharmaceutically acceptable derivative thereof provided herein, which iseffective for preventing TTR misfolding, or for treatment, prevention oramelioration of one or more symptoms of diseases or disorders associatedwith TTR misfolding, or diseases or disorders in which TTR misfolding,is implicated, within the packaging material, and a label that indicatesthat the compound or composition, or pharmaceutically acceptablederivative thereof, is used for modulating TTR folding, or fortreatment, prevention or amelioration of one or more symptoms ofdiseases or disorders associated with TTR misfolding, or diseases ordisorders in which TTR misfolding is implicated, are also provided.

DETAILED DESCRIPTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications are incorporated byreference in their entirety. In the event that there are a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, transthyretin or TTR is a 55 kDa homotetramercharacterized by 2,2,2 symmetry, having two identical funnel-shapedbinding sites at the dimer-dimer interface, where thyroid hormone (T4)can bind in blood plasma and CSF. TTR is typically bound to less than 1equiv of holo retinol binding protein. TTR is a 127-residue protein thattetramerizes under physiological conditions. TTR serves as the tertiarytransporter of thyroxine in the serum and the primary carrier in thecerebrospinal fluid. TTR also transports retinol through its associationwith retinol binding protein. TTR forms amyloid at low pH.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugsthereof. Such derivatives may be readily prepared by those of skill inthis art using known methods for such derivatization. The compoundsproduced may be administered to animals or humans without substantialtoxic effects and either are pharmaceutically active or are prodrugs.Pharmaceutically acceptable salts include, but are not limited to, aminesalts, such as but not limited to N,N′-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Other pharmaceuticallyacceptable salts include acid salts such as acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate;base salts including ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, basic nitrogen-containing groupscan be quaternized with such agents as lower alkyl halides, such asmethyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides, such as benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained. Pharmaceutically acceptable esters include, but arenot limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups,including, but not limited to, carboxylic acids, phosphoric acids,phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.Pharmaceutically acceptable enol ethers include, but are not limited to,derivatives of formula C═C(OR) where R is hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl orheterocyclyl. Pharmaceutically acceptable enol esters include, but arenot limited to, derivatives of formula C═C(OC(O)R) where R is hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl,cycloalkyl or heterocyclyl. Pharmaceutically acceptable solvates andhydrates are complexes of a compound with one or more solvent or watermolecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or4, solvent or water molecules.

As used herein, treatment means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use for treating TTR mediateddiseases or disorders, or diseases or disorders in which TTR, includingTTR misfolding, is implicated.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular compound or pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

As used herein, IC₅₀ refers to an amount, concentration or dosage of aparticular test compound that achieves a 50% inhibition of a maximalresponse, such as inhibition of TTR misfolding, in an assay thatmeasures such response.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized by one or more steps or processes orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, thepharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392).

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. In the case of amino acid residues, suchresidues may be of either the L- or D-form. The configuration fornaturally occurring amino acid residues is generally L. When notspecified the residue is the L form. As used herein, the term “aminoacid” refers to α-amino acids which are racemic, or of either the D- orL-configuration. The designation “d” preceding an amino acid designation(e.g., dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid.The designation “dl” preceding an amino acid designation (e.g., dlPip)refers to a mixture of the L- and D-isomers of the amino acid. It is tobe understood that the chiral centers of the compounds provided hereinmay undergo epimerization in vivo. As such, one of skill in the art willrecognize that administration of a compound in its (R) form isequivalent, for compounds that undergo epimerization in vivo, toadministration of the compound in its (S) form.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis, high performance liquid chromatography (HPLC) and massspectrometry (MS), used by those of skill in the art to assess suchpurity, or sufficiently pure such that further purification would notdetectably alter the physical and chemical properties, such as enzymaticand biological activities, of the substance. Methods for purification ofthe compounds to produce substantially chemically pure compounds areknown to those of skill in the art. A substantially chemically purecompound may, however, be a mixture of stereoisomers. In such instances,further purification might increase the specific activity of thecompound.

As used herein, alkyl, alkenyl and alkynyl carbon chains, if notspecified, contain from 1 to 20 carbons, or 1 or 2 to 16 carbons, andare straight or branched. Alkenyl carbon chains of from 2 to 20 carbons,in certain embodiments, contain 1 to 8 double bonds and alkenyl carbonchains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 doublebonds. Alkynyl carbon chains of from 2 to 20 carbons, in certainembodiments, contain 1 to 8 triple bonds, and the alkynyl carbon chainsof 2 to 16 carbons, in certain embodiments, contain 1 to 5 triple bonds.Exemplary alkyl, alkenyl and alkynyl groups herein include, but are notlimited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl,sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl,allyl (propenyl) and propargyl (propynyl). As used herein, lower alkyl,lower alkenyl, and lower alkynyl refer to carbon chains having fromabout 1 or about 2 carbons up to about 6 carbons. As used herein,“alk(en)(yn)yl” refers to an alkyl group containing at least one doublebond and at least one triple bond.

As used herein, “cycloalkyl” refers to a saturated mono- or multi-cyclic ring system, in certain embodiments of 3 to 10 carbon atoms, inother embodiments of 3 to 6 carbon atoms; cycloalkenyl and cycloalkynylrefer to mono- or multicyclic ring systems that respectively include atleast one double bond and at least one triple bond. Cycloalkenyl andcycloalkynyl groups may, in certain embodiments, contain 3 to 10 carbonatoms, with cycloalkenyl groups, in further embodiments, containing 4 to7 carbon atoms and cycloalkynyl groups, in further embodiments,containing 8 to 10 carbon atoms. The ring systems of the cycloalkyl,cycloalkenyl and cycloalkynyl groups may be composed of one ring or twoor more rings which may be joined together in a fused, bridged orspiro-connected fashion. “Cycloalk(en)(yn)yl” refers to a cycloalkylgroup containing at least one double bond and at least one triple bond.

As used herein, “aryl” refers to aromatic monocyclic or multicyclicgroups containing from 6 to 19 carbon atoms. Aryl groups include, butare not limited to groups such as unsubstituted or substitutedfluorenyl, unsubstituted or substituted phenyl, and unsubstituted orsubstituted naphthyl.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system, in certain embodiments, of about 5 to about 15members where one or more, in one embodiment 1 to 3, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. The heteroarylgroup may be optionally fused to a benzene ring. Heteroaryl groupsinclude, but are not limited to, furyl, imidazolyl, pyrimidinyl,tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl,oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl.

As used herein, a “heteroarylium” group is a heteroaryl group that ispositively charged on one or more of the heteroatoms.

As used herein, “heterocyclyl” refers to a monocyclic or multicyclicnon-aromatic ring system, in one embodiment of 3 to 10 members, inanother embodiment of 4 to 7 members, in a further embodiment of 5 to 6members, where one or more, in certain embodiments, 1 to 3, of the atomsin the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen isoptionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternizedto form an ammonium group where the substituents are selected as above.

As used herein, “aralkyl” refers to an alkyl group in which one of thehydrogen atoms of the alkyl is replaced by an aryl group.

As used herein, “heteroaralkyl” refers to an alkyl group in which one ofthe hydrogen atoms of the alkyl is replaced by a heteroaryl group.

As used herein, “halo”, “halogen” or “halide” refers to F, Cl, Br or I.

As used herein, pseudohalides or pseudohalo groups are groups thatbehave substantially similar to halides. Such compounds can be used inthe same manner and treated in the same manner as halides. Pseudohalidesinclude, but are not limited to, cyanide, cyanate, thiocyanate,selenocyanate, trifluoromethoxy, and azide.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by halogen. Such groups include,but are not limited to, chloromethyl, trifluoromethyland1-chloro-2-fluoroethyl.

As used herein, “haloalkoxy” refers to RO— in which R is a haloalkylgroup.

As used herein, “sulfinyl” or “thionyl” refers to —S(O)—. As usedherein, “sulfonyl” or “sulfuryl” refers to —S(O)2-. As used herein,“sulfo” refers to —S(O)2O—.

As used herein, “carboxy” refers to a divalent radical, —C(O)O—.

As used herein, “aminocarbonyl” refers to —C(O)NH2.

As used herein, “alkylaminocarbonyl” refers to —C(O)NHR in which R isalkyl, including lower alkyl. As used herein, “dialkylaminocarbonyl”refers to —C(O)NR′R in which R′ and R are independently alkyl, includinglower alkyl; “carboxamide” refers to groups of formula —NR′COR in whichR′ and R are independently alkyl, including lower alkyl.

As used herein, “diarylaminocarbonyl” refers to —C(O)NRR′ in which R andR′ are independently selected from aryl, including lower aryl, such asphenyl.

As used herein, “arylalkylaminocarbonyl” refers to —C(O)NRR′ in whichone of R and R′ is aryl, including lower aryl, such as phenyl, and theother of R and R′ is alkyl, including lower alkyl.

As used herein, “arylaminocarbonyl” refers to —C(O)NHR in which R isaryl, including lower aryl, such as phenyl.

As used herein, “hydroxycarbonyl” refers to —COOH.

As used herein, “alkoxycarbonyl” refers to —C(O)OR in which R is alkyl,including lower alkyl.

As used herein, “aryloxycarbonyl” refers to —C(O)OR in which R is aryl,including lower aryl, such as phenyl.

As used herein, “alkoxy” and “alkylthio” refer to RO— and RS—, in whichR is alkyl, including lower alkyl.

As used herein, “aryloxy” and “arylthio” refer to RO— and RS—, in whichR is aryl, including lower aryl, such as phenyl.

As used herein, “alkylene” refers to a straight, branched or cyclic, incertain embodiments straight or branched, divalent aliphatic hydrocarbongroup, in one embodiment having from 1 to about 20 carbon atoms, inanother embodiment having from 1 to 12 carbons. In a further embodimentalkylene includes lower alkylene. There may be optionally inserted alongthe alkylene group one or more oxygen, sulfur, including S(═O) andS(═O)2 groups, or substituted or unsubstituted nitrogen atoms, including—NR— and —N+RR— groups, where the nitrogen substituent(s) is(are) alkyl,aryl, aralkyl, heteroaryl, heteroaralkyl or COR′, where R′ is alkyl,aryl, aralkyl, heteroaryl, heteroaralkyl, —OY or —NYY, where Y ishydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. Alkylenegroups include, but are not limited to, methylene (—CH2-), ethylene(—CH2CH2-), propylene (—(CH2)3-), methylenedioxy (—O—CH2-O—) andethylenedioxy (—O—(CH2)2-O—). The term “lower alkylene” refers toalkylene groups having 1 to 6 carbons. In certain embodiments, alkylenegroups are lower alkylene, including alkylene of 1 to 3 carbon atoms.

As used herein, “azaalkylene” refers to —(CRR)n-NR—(CRR)m-, where n andm are each independently an integer from 0 to 4. As used herein,“oxaalkylene” refers to —(CRR)n-O—(CRR)m-, where n and m are eachindependently an integer from 0 to 4. As used herein, “thiaalkylene”refers to —(CRR)n-S—(CRR)m-, —(CRR)n-S(═O)—(CRR)m-, and—(CRR)n-S(═O)2-(CRR)m-, where n and m are each independently an integerfrom 0 to 4.

As used herein, “alkenylene” refers to a straight, branched or cyclic,in one embodiment straight or branched, divalent aliphatic hydrocarbongroup, in certain embodiments having from 2 to about 20 carbon atoms andat least one double bond, in other embodiments 1 to 12 carbons. Infurther embodiments, alkenylene groups include lower alkenylene. Theremay be optionally inserted along the alkenylene group one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, where thenitrogen substituent is alkyl. Alkenylene groups include, but are notlimited to, —CH═CH—CH═CH— and —CH═CH—CH2-. The term “lower alkenylene”refers to alkenylene groups having 2 to 6 carbons. In certainembodiments, alkenylene groups are lower alkenylene, includingalkenylene of 3 to 4 carbon atoms.

As used herein, “alkynylene” refers to a straight, branched or cyclic,in certain embodiments straight or branched, divalent aliphatichydrocarbon group, in one embodiment having from 2 to about 20 carbonatoms and at least one triple bond, in another embodiment 1 to 12carbons. In a further embodiment, alkynylene includes lower alkynylene.There may be optionally inserted along the alkynylene group one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, where thenitrogen substituent is alkyl. Alkynylene groups include, but are notlimited to, —C≡C—C≡C—, —C≡C— and —C≡C—CH2-. The term “lower alkynylene”refers to alkynylene groups having 2 to 6 carbons. In certainembodiments, alkynylene groups are lower alkynylene, includingalkynylene of 3 to 4 carbon atoms.

As used herein, “alk(en)(yn)ylene” refers to a straight, branched orcyclic, in certain embodiments straight or branched, divalent aliphatichydrocarbon group, in one embodiment having from 2 to about 20 carbonatoms and at least one triple bond, and at least one double bond; inanother embodiment 1 to 12 carbons. In further embodiments,alk(en)(yn)ylene includes lower alk(en)(yn)ylene. There may beoptionally inserted along the alkynylene group one or more oxygen,sulfur or substituted or unsubstituted nitrogen atoms, where thenitrogen substituent is alkyl. Alk(en)(yn)ylene groups include, but arenot limited to, —C═C—(CH2)n-C≡C—, where n is 1 or 2. The term “loweralk(en)(yn)ylene” refers to alk(en)(yn)ylene groups having up to 6carbons. In certain embodiments, alk(en)(yn)ylene groups have about 4carbon atoms.

As used herein, “cycloalkylene” refers to a divalent saturated mono- ormulticyclic ring system, in certain embodiments of 3 to 10 carbon atoms,in other embodiments 3 to 6 carbon atoms; cycloalkenylene andcycloalkynylene refer to divalent mono- or multicyclic ring systems thatrespectively include at least one double bond and at least one triplebond. Cycloalkenylene and cycloalkynylene groups may, in certainembodiments, contain 3 to 10 carbon atoms, with cycloalkenylene groupsin certain embodiments containing 4 to 7 carbon atoms andcycloalkynylene groups in certain embodiments containing 8 to 10 carbonatoms. The ring systems of the cycloalkylene, cycloalkenylene andcycloalkynylene groups may be composed of one ring or two or more ringswhich may be joined together in a fused, bridged or spiro-connectedfashion. “Cycloalk(en)(yn)ylene” refers to a cycloalkylene groupcontaining at least one double bond and at least one triple bond.

As used herein, “arylene” refers to a monocyclic or polycyclic, incertain embodiments monocyclic, divalent aromatic group, in oneembodiment having from 5 to about 20 carbon atoms and at least onearomatic ring, in another embodiment 5 to 12 carbons. In furtherembodiments, arylene includes lower arylene. Arylene groups include, butare not limited to, 1,2-, 1,3- and 1,4-phenylene. The term “lowerarylene” refers to arylene groups having 6 carbons.

As used herein, “heteroarylene” refers to a divalent monocyclic ormulticyclic aromatic ring system, in one embodiment of about 5 to about15 atoms in the ring(s), where one or more, in certain embodiments 1 to3, of the atoms in the ring system is a heteroatom, that is, an elementother than carbon, including but not limited to, nitrogen, oxygen orsulfur. The term “lower heteroarylene” refers to heteroarylene groupshaving 5 or 6 atoms in the ring.

As used herein, “heterocyclylene” refers to a divalent monocyclic ormulticyclic non-aromatic ring system, in certain embodiments of 3 to 10members, in one embodiment 4 to 7 members, in another embodiment 5 to 6members, where one or more, including 1 to 3, of the atoms in the ringsystem is a heteroatom, that is, an element other than carbon, includingbut not limited to, nitrogen, oxygen or sulfur.

As used herein, “substituted alkyl,” “substituted alkenyl,” “substitutedalkynyl,” “substituted cycloalkyl,” “substituted cycloalkenyl,”“substituted cycloalkynyl,” “substituted aryl,” “substitutedheteroaryl,” “substituted heterocyclyl,” “substituted alkylene,”“substituted alkenylene,” “substituted alkynylene,” “substitutedcycloalkylene,” “substituted cycloalkenylene,” “substitutedcycloalkynylene,” “substituted arylene,” “substituted heteroarylene” and“substituted heterocyclylene” refer to alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl,alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene,cycloalkynylene, arylene, heteroarylene and heterocyclylene groups,respectively, that are substituted with one or more substituents, incertain embodiments one, two, three or four substituents, where thesubstituents are as defined herein, in one embodiment selected from Q1.

As used herein, “alkylidene” refers to a divalent group, such as ═CR′R″,which is attached to one atom of another group, forming a double bond.Alkylidene groups include, but are not limited to, methylidene (═CH2)and ethylidene (═CHCH3). As used herein, “arylalkylidene” refers to analkylidene group in which either R′ or R″ is an aryl group.“Cycloalkylidene” groups are those where R′ and R″ are linked to form acarbocyclic ring. “Heterocyclylidene” groups are those where at leastone of R′ and R″ contain a heteroatom in the chain, and R′ and R″ arelinked to form a heterocyclic ring.

As used herein, “amido” refers to the divalent group —C(O)NH—.“Thioamido” refers to the divalent group —C(S)NH—. “Oxyamido” refers tothe divalent group —OC(O)NH—. “Thiaamido” refers to the divalent group—SC(O)NH—. “Dithiaamido” refers to the divalent group —SC(S)NH—.“Ureido” refers to the divalent group —HNC(O)NH—. “Thioureido” refers tothe divalent group —HNC(S)NH—.

As used herein, “semicarbazide” refers to —NHC(O)NHNH—. “Carbazate”refers to the divalent group —OC(O)NHNH—. “Isothiocarbazate” refers tothe divalent group —SC(O)NHNH—. “Thiocarbazate” refers to the divalentgroup —OC(S)NHNH—. “Sulfonylhydrazide” refers to the divalent group—SO2NHNH—. “Hydrazide” refers to the divalent group —C(O)NHNH—. “Azo”refers to the divalent group —N═N—. “Hydrazinyl” refers to the divalentgroup —NH—NH—.

Where the number of any given substituent is not specified (e.g.,haloalkyl), there may be one or more substituents present. For example,“haloalkyl” may include one or more of the same or different halogens.As another example, “C1-3alkoxyphenyl” may include one or more of thesame or different alkoxy groups containing one, two or three carbons.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

B. TTR and Amyloid Disease

At least some amyloid diseases appear to be caused by the deposition ofany one of more than 20 nonhomologous proteins or protein fragments,ultimately affording a fibrillar cross-β-sheet quaternary structure.Formation of amyloid fibrils from a normally folded protein liketransthyretin requires protein misfolding to produce anassembly-competent intermediate. The process of transthyretin (TTR)amyloidogenesis appears to cause three different amyloid diseases—senilesystemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) andfamilial amyloid cardiomyopathy (FAC). SSA is associated with thedeposition of wild-type TTR, while FAP and FAC are caused by theamyloidogenesis of one of over 80 TTR variants. See, for example, Colon,W.; Kelly, J. W. Biochemistry 1992, 31, 8654-60; Kelly, J. W. Curr.Opin. Struct. Biol. 1996, 6, 11-7; Liu, K.; et al. Nat. Struct. Biol.2000, 7, 754-7; Westermark, P.; et al. Proc. Natl. Acad. Sci. U.S.A.1990, 87, 2843-5; Saraiva, M. J.; et al. J. Clin. Invest. 1985, 76,2171-7; Jacobson, D. R.; et al. N. Engl. J. Med. 1997, 336, 466-73;Buxbaum, J. N.; Tagoe, C. E. Ann. Rev. Med. 2000, 51, 543-569; andSaraiva, M. J. Hum. Mutat. 1995, 5, 191-6, each of which is incorporatedby reference in its entirety.

TTR is a 55 kDa homotetramer characterized by 2,2,2 symmetry, having twoidentical funnel-shaped binding sites at the dimer-dimer interface,where thyroid hormone (T4) can bind in blood plasma and CSF. TTR istypically bound to less than 1 equiv of holo retinol binding protein.TTR misfolding including tetramer dissociation into monomers followed bytertiary structural changes within the monomer render the proteincapable of misassembly, ultimately affording amyloid. The availabletreatment for FAP employs gene therapy mediated by liver transplantationto replace variant TTR in the blood with the wild type (WT) protein.This treatment is not applicable to most patients with FAC or SSAbecause of most are over the age of 60 and are not candidates for livertransplantation due to their health status and impaired cardiacfunction. Furthermore, for several TTR variants associated with FAP,progressive cardiac amyloidosis developed after liver transplantationwith wt TTR deposition in cardiac tissues, leading to death. Livertransplantation therapy would also fail for approximately 10 of the TTRvariants that deposit amyloid fibrils in the leptomeninges leading toCNS disease, as this TTR is synthesized by the choroid plexus. Hence, itis desirable to develop a general noninvasive drug-based therapeuticstrategy. It can be desirable for the drug to be non-protein,non-peptide, or non-nucleic acid based. See, for example, Blake, C. C.;et al. J. Mol. Biol. 1978, 121, 339-56; Wojtczak, A.; et al. ActaCrystallogr., Sect. D 1996, 758-810; Monaco, H. L.; Rizzi, M.; Coda, A.Science 1995, 268, 1039-41; Lai, Z.; Colon, W.; Kelly, J. W.Biochemistry 1996, 35, 6470-82; Holmgren, G.; et al. Lancet 1993, 341,1113-6; Suhr, O. B.; Ericzon, B. G.; Friman, S. Liver Transpl. 2002, 8,787-94; Dubrey, S. W.; et al. Transplantation 1997, 64, 74-80; Yazaki,M.; et al. Biochem. Biophys. Res. Commun. 2000, 274, 702-6; andCornwell, C. G. III; et al. Am. J. of Med. 1983, 75, 618-623, each ofwhich is incorporated by reference in its entirety.

C. Compounds

In one embodiment, the compounds for use in the compositions and methodsprovided herein have formulae I:

where where Y is COOH, tetrazolyl, CONHOH, B(OH)₂ or OH;

X¹ is O, —O—N═CH— or NH;

R⁴ is hydrogen, halo, OH, or alkyl; and

R¹, R² and R³ are each independently selected from hydrogen, halo, OH,B(OH)₂ or CF₃.

In another embodiment, the compounds have formulae I, where Y is COOH.In another embodiment, Y is tetrazolyl, B(OH)₂ or CONHOH. In anotherembodiment, Y is OH.

In another embodiment, X¹ is O or NH. In another embodiment, X¹ is—O—N═CH—.

In another embodiment, the compounds have formulae I, where R¹, R² andR³ are each independently selected from hydrogen, halo and OH. Inanother embodiment, the compounds have formulae I, where R¹, R² and R³are each independently selected from hydrogen and CF₃. In anotherembodiment, the compounds have formulae I, where R¹ and R² are eachindependently hydrogen and R³ is CF₃. In another embodiment, thecompounds have formulae I, where R¹, R² and R³ are each independentlyselected from hydrogen, halo and B(OH)₂. In another embodiment, thecompounds have formulae I, where R¹, R² and R³ are each independentlyselected from hydrogen, Br and I. In another embodiment, the compoundshave formulae I, where R¹, R² and R³ are each independently selectedfrom halo and OH. In another embodiment, the compounds have formulae I,where R¹, R² and R³ are each independently selected from halo andB(OH)₂. In another embodiment, the compounds have formulae I, where R¹and R² are each halo and R³ is OH. In another embodiment, the compoundshave formulae I, where R¹ and R² are each halo and R³ is B(OH)₂. Inanother embodiment, the compounds have formulae I, where R¹ and R² areeach halo and R³ is H.

In another embodiment, R⁴ is hydrogen or OH. In another embodiment, R⁴is hydrogen. In another embodiment, R⁴ is OH.

In another embodiment, the compound has formulae I, and is selectedfrom:

D. Preparation of the Compounds

The compounds provided herein may be made by the methods shown below andin the Examples, or by other methods well known to those of skill in theart. Starting materials in these synthetic methods may be obtained fromcommercial sources (e.g., Aldrich Chemical Co., Milwaukee, Wis., USA).

Reagents and solvents were purchased from Aldrich, Lancaster, Acros,Combi-Blocks, Matrix and Pfaltz-Bauer. THF and CH₂Cl₂ were dried bypassage over Al₂O₃. Other solvents and reagents were obtained fromcommercial suppliers and were used without further purification unlessotherwise noted. Reactions were monitored by analytical thin layerchromatography (TLC) on silica gel 60 F₂₅₄ pre-coated plates withfluorescent indicator purchased from EM Science. Visualization of theTLC plates was accomplished by UV illumination, phosphomolybdic acidtreatment followed by heat or ceric ammonium molybdate treatmentfollowed by heat. Flash chromatography was performed using silica gel 60(230-400 mesh) from EM Science. The purity of new compounds that wereessential to the conclusions drawn in the text were determined by HPLC.Normal phase HPLC was performed with a Waters 600 pump/controller, aWaters 996 photodiode array detector and a Waters NovaPak silica column.The solvent system employed was hexanes and ethyl acetate, and gradientswere run from 50:50 hexanes:ethyl acetate to 0:100 hexanes:ethyl acetateover 30 min. Reverse phase HPLC was performed with a Waters 600pump/controller, a Waters 2487 dual wavelength detector and a Vydacprotein and peptide C18 column. Solvent system A was 95:5water:acetonitrile with 0.5% trifluoroacetic acid and solvent B was 5:95water:acetonitrile with 0.5% trifluoroacetic acid. Gradients were runfrom 100:0 A:B to 0:100 A:B over 20 min with a hold at 100% B for anadditional 10 min. Circular dichroism spectroscopy was performed on anAVIV Instruments spectrometer, model 202SF. NMR spectra were recorded ona Varian FT NMR spectrometer at a proton frequency of 400 MHz. Protonchemical shifts are reported in parts per million (ppm) with referenceto CHCl₃ as the internal chemical shift standard (7.26 ppm) unlessotherwise noted. Coupling constants are reported in hertz (Hz). Carbonchemical shifts are reported in parts per million (ppm) with referenceto CDCl₃ as the chemical shift standard (77.23 ppm) unless otherwisenoted. All mass spectra were obtained at The Scripps Research InstituteCenter for Mass Spectrometry or the University of Illinois MassSpectrometry Laboratory.

In general, the compounds can be synthesized by methods known in theart. One method of making the compounds is a Suzuki coupling:

For example, a biphenyl compound can be formed by a Suzuki coupling of aphenyl boronic acid with a bromobenzene or an iodobenzene. Appropriateprotecting groups may be needed to avoid forming side products duringthe preparation of a compound. For example, an amino substituent can beprotected by a suitable amino protecting group such as trifluoroacetylor tert-butoxycarbonyl. Other protecting groups and reaction conditionscan be found in T. W. Greene, Protective Groups in Organic Synthesis,(3rd, 1999, John Wiley & Sons, New York, N.Y.).

Certain of the compounds provided herein were synthesized using aPd-mediated Suzuki coupling between an aryl halide and an aryl boronicacid. For example, the synthesis of certain compounds was achieved byacetylation of the appropriate phenol with acetic anhydride andpyridine, followed by Suzuki coupling with the appropriate phenylboronic acid under the standard Suzuki coupling reaction conditions.Removal of the ester with Na⁰ and MeOH (Zemplén conditions) provided thedesired biphenyl phenols. See, for example, Miyaura, N.; Yanagi, T.;Suzuki, A. Synth. Commun. 1981, 11, 513-519; Sharp, M. J.; Snieckus, V.Tetrahedron Lett. 1985, 26, 5997-6000; Sharp, M. J.; Cheng, W.;Snieckus, V. Tetrahedron Lett. 1987, 28, 5093-5096; Pozsgay, V.; Nanasi,P.; Neszmelyi, A. Carbohydr. Res. 1981, 90, 215-231; Jendralla, H.;Chen, L.-J. Synthesis 1990, 827-833; and Kelm, J.; Strauss, K.Spectrochim. Acta, Part A 1981, 37, 689-692, each of which isincorporated by reference in its entirety

Other compounds provided herein were synthesized using solid-phasemethods. The appropriate benzoic acid was coupled to Wang resin via anester linkage, affording a resin-bound phenyliodide, which was thencoupled to a phenyl boronic acid, and cleaved from the resin with a 1:1mixture of TFA:CH₂Cl₂. See, for example, Guiles, J. W.; Johnson, S. G.;Murray, W. V. J. Org. Chem. 1996, 61, 5169-5171, which is incorporatedby reference in its entirety.

Carboxylate-containing compounds were assembled by coupling of abenzoate ester with the appropriate fluorophenyl boronic acid utilizingstandard Suzuki coupling conditions. The ester was then saponified withLiOH.H₂O to provide the corresponding carboxylate. See, for example,Bumagin, N. A.; Bykov, V. V. Tetrahedron 1997, 53, 14437-14450;Ananthakrishnanadar, P.; Kannan, N. J. Chem. Soc., Perkin Trans. 2 1982,1305-1308; Homsi, F.; Nozaki, K.; Hiyama, T. Tetrahedron Lett. 2000, 41,5869-5872; and Hajduk, P. J.; et al. J. Med. Chem. 1997, 40, 3144-3150,each of which is incorporated by reference in its entirety.

3′,5′-Dihalo-4′-hydroxyl-containing analogs were synthesized by firstprotecting the commercially available bromophenol as the methyl ether(MeI and K₂CO₃). Suzuki coupling with a (methoxycarbonylphenyl) boronicacid resulted in, the formation of the fully protected biphenylsubstrates. BBr₃-mediated methyl ether cleavage and saponification withLiOH.H₂O provided the fully functionalized compounds.

A series of halogenated biphenyls were assembled by Suzuki coupling ofiodobenzene with a series of halogen-containing boronic acids. See, forexample, Patrick, T. B.; Willaredt, R. P. DeGonia, D. J. J. Org. Chem.1985, 50, 2232-2235; Kuchar, M.; et al. Collection of CzechoslovakChemical Communications 1988, 53, 1862-1872; Allen, K. J.; Bolton, R.;Williams, G. H. J. Chem. Soc., Perkin Trans. 2 1983, 691-695; Nakada,M.; et al. Bull. Chem. Soc. Jpn. 1989, 62, 3122-3126; and Weingarten, H.J. Org. Chem. 1961, 26, 730-733, each of which is incorporated byreference in its entirety.

E. Formulation of Pharmaceutical Compositions

The pharmaceutical compositions provided herein contain therapeuticallyeffective amounts of one or more of the compounds provided herein thatare useful in the prevention, treatment, or amelioration of one or moreof the symptoms of diseases or disorders associated with transthyretin(TTR) misfolding, or in which TTR misfolding is implicated, in apharmaceutically acceptable carrier. Diseases or disorders associatedwith TTR misfolding include, but are not limited to, familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, senile systemicamyloidosis, Alzheimer's disease, spongiform encephalopathy (CreutzfeldtJakob disease), polyneuropathy, type II diabetes, medullary carcinoma ofthe thyroid and obesity. Pharmaceutical carriers suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

The compositions contain one or more compounds provided herein. Thecompounds are, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, thecompounds described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof is (are) mixed with asuitable pharmaceutical carrier. The compounds may be derivatized as thecorresponding salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs prior to formulation, as described above. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates oneor more of the symptoms of diseases or disorders associated with TTRmisfolding or in which TTR misfolding is implicated.

In one embodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described and thenextrapolated therefrom for dosages for humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to ameliorate one or more of the symptoms ofdiseases or disorders associated with TTR misfolding or in which TTRmisfolding is implicated, as described herein.

In one embodiment, a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.1 ng/ml toabout 50-100 μg/ml. The pharmaceutical compositions, in anotherembodiment, should provide a dosage of from about 0.001 mg to about 2000mg of compound per kilogram of body weight per day. Pharmaceuticaldosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment fromabout 10 mg to about 500 mg of the active ingredient or a combination ofessential ingredients per dosage unit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as TWEEN®, or dissolution in aqueous sodium bicarbonate.Derivatives of the compounds, such as prodrugs of the compounds may alsobe used in formulating effective pharmaceutical compositions.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

1. Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

a. Solid Compositions for Oral Administration

In certain embodiments, the formulations are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder; a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an emetic coating; and a film coating.Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, molasses,polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The compound, or pharmaceutically acceptable derivative thereof, couldbe provided in a composition that protects it from the acidicenvironment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. Thecomposition may also be formulated in combination with an antacid orother such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

b. Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g., for example, in a polyethylene glycol, may be dilutedwith a sufficient quantity of a pharmaceutically acceptable liquidcarrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

2. Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a compound providedherein is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEENâ 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active compound to thetreated tissue(s).

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

3. Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

4. Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01% - 10% isotonic solutions, pH about 5-7, withappropriate salts.

5. Compositions for Other Routes of Administration

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and buccal and rectaladministration, are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and5,860,957.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

6. Targeted Formulations

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLV's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

7. Articles of Manufacture

The compounds or pharmaceutically acceptable derivatives may be packagedas articles of manufacture containing packaging material, a compound orpharmaceutically acceptable derivative thereof provided herein, which iseffective for modulating TTR folding, or for treatment, prevention oramelioration of one or more symptoms of TTR mediated diseases ordisorders, or diseases or disorders in which TTR misfolding, isimplicated, within the packaging material, and a label that indicatesthat the compound or composition, or pharmaceutically acceptablederivative thereof, is used for modulating TTR folding, or fortreatment, prevention or amelioration of one or more symptoms of TTRmediated diseases or disorders, or diseases or disorders in which TTRmisfolding is implicated.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment. A wide array of formulations ofthe compounds and compositions provided herein are contemplated as are avariety of treatments for any disease or disorder in which TTRmisfolding is implicated as a mediator or contributor to the symptoms orcause.

F. Evaluation of the Activity of the Compounds

A number of in vitro tests can be used to evaluate the compounds fortheir ability to stabilize transthyretin tetramers or prevent formationof fibrils. The tests can include a fibril formation assay, a plasmaselectivity assay, determination of the three-dimensional structure of atransthyretin:compound complex (e.g., by X-ray crystallography),kinetics of transthyretin tetramer dissociation or fibril formations,and determining the stoichiometry and energetics oftransthyretin:compound interactions, by, for example, centrifugation orcalorimetry. Details of exemplary in vitro assays are presented in theExamples.

The transthyretin used in the screening methods can be wild typetransthyretin or a mutant transthyretin, such as a naturally occurringmutant transthyretin causally associated with the incidence of atransthyretin amyloid disease such as familial amyloid polyneuropathy orfamilial amyloid cardiomyopathy. Exemplary naturally occurring mutanttransthyretins include, but are not limited to, V1221, V30M, L55P (themutant nomenclature describes the substitution at a recited amino acidposition, relative to the wild type; see, e.g., Saraiva et al. (2001)Hum. Mut. 17:493-503).

For a compound to be an effective drug against TTR amyloidosis, it hasto bind to TTR strongly and selectively, so that in blood plasma itpartitions into TTR in the presence of all of the other plasma proteins.Therefore, two assays were used to evaluate the compounds providedherein. The first assay was a stagnant fibril formation assay that wehave described in depth previously. In this assay, a test compound isadded at 3.6 or 7.2 μM to a solution of TTR at 3.6 μM. At these twoconcentrations there is enough test compound to load either one or bothof TTR's binding sites. The solution is then placed under amyloidogenicconditions by lowering the pH to 4.4 (the pH at which the rate of TTRamyloid formation is maximal). After 72 h, the turbidity of the TTRsolutions (which is related to the extent of TTR aggregation) with thetest compounds (T_(test)) is measured and compared to that of a solutionthat lacks any test compound (T_(control)). The extent of inhibition offibril formation is calculated from the differences in turbidity withand without the test compound as:Inhibition=(T _(control) −T _(test))/(T _(control))×100%

High inhibition values indicate very active compounds.

The second assay was an antibody capture method recently developed bythis laboratory to measure the test compounds' abilities to bind to TTRin human blood plasma in the presence of all of the other plasmaproteins. In this assay the test compound is dissolved to 10.8 μM inhuman blood plasma (about 3 times the concentration of TTR) andincubated for 24 h. The TTR and any bound small molecule is thenimmunoprecipitated using a polyclonal TTR antibody bound to sepharoseresin. After washing the resin, the antibody-TTR complex is dissociatedat high pH and the stoichiometry of TTR to test compound is determinedfrom their peak areas in an HPLC.

G. Methods of Use of the Compounds and Compositions

Also provided are methods for the stabilization of transthyretin in atissue or in a biological fluid, and thereby inhibiting misfolding.Generally, the method involves administering to the tissue or biologicalfluid a stabilizing amount of a compound provided herein that binds totransthyretin and prevents dissociation of the transthyretin tetramer bykinetic stabilization of the native state of the transthyretin tetramer.

Thus, methods which stabilize transthyretin in a diseased tissueameliorate misfolding and lessen symptoms of an associated disease and,depending upon the disease, can contribute to cure of the disease. Alsocontemplated herein is inhibition of transthyretin misfolding in atissue and/or within a cell. The extent of misfolding, and therefore theextent of inhibition achieved by the present methods, can be evaluatedby a variety of methods, such as are described in the Examples and ininternational patent application publication no. WO2004/056315. Thedisclosure of the above-referenced application is incorporated herein byreference in its entirety.

Also provided herein is a method of treating, preventing, orameliorating one or more symptoms of a transthyretin amyloid disease,the method involving administering a therapeutically effective amount ofa compound provided herein. In one embodiment, the compound preventsdissociation of a transthyretin tetramer by kinetic stabilization of thenative state of the transthyretin tetramer. The transthyretin amyloiddisease can be, for example, familial amyloid polyneuropathy, familialamyloid cardiomyopathy, or senile systemic amyloidosis. Othertransthyretin amyloid diseases include but are not limited toAlzheimer's disease, spongiform encephalopathy (Creutzfeldt Jakobdisease), polyneuropathy, type II diabetes and medullary carcinoma ofthe thyroid (see, e.g., International Patent Application PublicationNos. WO 98/27972 and WO 95/12815).

Methods of treating, preventing, or ameliorating one or more symptoms ofa transthyretin mediated disease or disorder by administering a compoundprovided herein are provided. Transthyretin mediated diseases anddisorders include but are not limited to obesity (see, e.g.,International Patent Application Publication No. WO 02/059621).

Further provided is a method of stabilizing TTR tetramers using acompound or composition provided herein. Also provided is a method ofinhibiting formation of TTR amyloid using a compound or compositionprovided herein.

Also provided herein is use of any of the compounds or pharmaceuticalcompositions described herein for the treatment, prevention, oramelioration of one or more symptoms of a transthyretin amyloid disease(e.g., familial amyloid polyneuropathy, familial amyloid cardiomyopathy,or senile systemic amyloidosis).

Also provided herein is use of any of the compounds or pharmaceuticalcompositions described herein in the manufacture of a medicament for thetreatment, prevention, or amelioration of one or more symptoms of atransthyretin amyloid disease (e.g., familial amyloid polyneuropathy,familial amyloid cardiomyopathy, or senile systemic amyloidosis).

H. Combination Therapy

The compounds and compositions provided herein may be administered as amonotherapy or in combination with other active ingredients. Forexample, the compounds and compositions may be administered incombination with other compounds known for the treatment of amyloidosesand amyloid disorders, including but not limited to, those disclosed inInternational Patent Application Publication Nos. WO 98/27972, WO02/059621, WO 95/12815, and WO2004/056315. Further active ingredientsfor combination therapy include but are not limited to ARICEPT® andother products approved for treatment of amyloidoses, including but notlimited to familial amyloid polyneuropathy, familial amyloidcardiomyopathy, or senile systemic amyloidosis, Alzheimer's disease,spongiform encephalopathy (Creutzfeldt Jakob disease), polyneuropathy,type II diabetes and medullary carcinoma of the thyroid. Further activeingredients for combination therapy include but are not limited toproducts approved for treatment of obesity.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1

The compounds provided herein were subjected to a stagnant fibrilformation assay. Compounds were dried over P₂O₅ overnight and dissolvedin DMSO to a final concentration of 7.2 mM to provide a primary stocksolution (10× stock). A secondary stock solution was prepared byfive-fold dilution of the primary stock solution with DMSO to a finalconcentration of 1.44 mM (2× stock). The acid-mediated amyloidogenicityof TTR (3.6 μM) in the presence of inhibitors (1.44 mM) was measured asfollows: To a disposable UV cuvette were added 495 μL of a 0.4 mg/mL WTTTR protein solution in 10 mM sodium phosphate, 100 mM KCl and 1 mM EDTA(pH 7.6) and 5 μL of the 1.44 mM secondary stock inhibitor solution inDMSO (2× stock). The mixture was vortexed and incubated for 30 min (25°C.), at which time the pH was lowered to 4.4 with 500 μL of 200 mMacetate, 100 mM KCl and 1 mM EDTA (pH 4.2). The final 1 mL solution wasvortexed and incubated for 72 h at 37° C. without agitation. After 72 h,the cuvettes were vortexed to suspend any fibrils present, and theturbidity of the suspension was measured at 350 and 400 nm using aUV-vis spectrometer. The percent fibril formation was obtained by theratio of the observed turbidities for each TTR plus inhibitor samplerelative to that of a sample prepared the same way, but lackinginhibitor, multiplied by 100. The fibril formation assay employingequimolar inhibitor and TTR concentrations (3.6 μM) was performed asabove using a 1× secondary stock solution. The 1× stock solution wasprepared by ten-fold dilution of the 7.2 mM 10× primary stock solutionwith DMSO to a final concentration of 0.72 mM and used in the fibrilformation assay as described above. All assays were performed intriplicate and all compounds were assayed using wild-type TTR. Allcompounds were found to be soluble throughout the course of theexperiment by testing the turbidities of the solutions in the absence ofWT TTR, ensuring that turbidity was the result of TTR amyloid formation.

The binding stoichiometries of potential inhibitors to TTR in bloodplasma were evaluated by an antibody capture/HPLC method. A 1.5-mLeppendorf tube was filled with 1.0 mL of human blood plasma and 7.5 μLof a 1.44 mM DMSO solution of the inhibitor under evaluation. Thesolution was incubated and gently rocked at 37° C. for 24 h. A 1:1gel:TSA (Tris saline) slurry (125 μL) of quenched sepharose was added tothe solution and gently rocked at 4° C. for 1 h. The solution wascentrifuged (16,000×g) and the supernatant was divided into two 400 μLaliquots, which were then added to different 200 μL samples of a 1:1gel:TSA slurry of the anti-TTR antibody-conjugated sepharose. Thesolutions were gently rocked at 4° C. for 20 min, centrifuged(16,000×g), and the supernatant was removed. The gel was washed with 1mL of TSA/0.05% saponin (3×, 10 min each) at 4° C., followed by 1 mL ofTSA (2×, 10 min each) at 4° C. The samples were centrifuged (16,000×g),the final wash was removed, and 155 μL of 100 mM triethylamine, pH 11.5,was added to elute the TTR and bound inhibitors from the antibodies.After gentle rocking at 4° C. for 30 min, the elution sample wascentrifuged (16,000×g) and 145μL of the supernatant, containing TTR andinhibitor, were removed. The supernatant was then analyzed byreverse-phase HPLC as described previously. See, for example, Purkey, H.E.; Dorrell, M. I.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,5566-71, which is incorporated by reference in its entirety.

The kinetics of TTR tetramer dissociation was evaluated by linkedmonomer unfolding in urea. Slow tetramer dissociation is not detectableby far-UV CD spectroscopy, but is linked to the rapid (500,000-foldfaster) unfolding step easily detectable by far-UV CD as describedpreviously. TTR tetramer (3.6 μM) dissociation kinetics as a function ofinhibitor (3.6 μM) were evaluated by adding 3.6 μL of a 1 mM solution(in ethanol) of the inhibitor of interest to 69 μL of WT TTR (2.90mg/mL, 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH 7.0) to whichwas added 127.4 μL of phosphate buffer. For an inhibitor concentration(7.2 μM) twice that of the TTR concentration (3.6 μM), 7.2 μL of a 1 mMsolution (in ethanol) of the inhibitor of interest was added to 69 μL ofWT TTR (2.90 mg/mL, 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH7.0) to which was added 123.8 μL of phosphate buffer. 100 μL of theprotein-inhibitor solution of interest was added to a solution of 600 μLof 10.3 M urea and 300 μL of phosphate buffer, to yield a final ureaconcentration of 6.5 M. The solutions were vortexed and the circulardichroism spectra were collected at the following intervals: 0, 5, 8,23, 46, 71, 95, 118, 144 and 168 h. A control sample containing 7.2 μLof ethanol rather than inhibitor was prepared for comparison and thespectra were collected at the time points identified above. CD spectrawere collected between 220 and 213 nm, with scanning every 0.5 nm and anaveraging time of 10 sec. Each wavelength was scanned once. The valuesfor the amplitude were averaged between 220 and 213 nm to determine theextent of β-sheet loss throughout the experiment.

The rate of acid-mediated fibril formation was followed at pH 4.4 byturbidity. Compounds were dried over P₂O₅ overnight and dissolved inDMSO to a final concentration of 7.2 mM to provide a primary stocksolution (10× stock). A secondary stock solution was prepared byfive-fold DMSO dilution of the primary stock solution to yield a finalconcentration of 1.44 mM (2× stock). The fibril formation assayemploying an inhibitor concentration of 7.2 μM relative to 3.6 μM TTR(tetramer) was performed as follows: To a disposable UV cuvette wereadded 495 μL of a 0.4 mg/mL WT TTR protein solution in 10 mM sodiumphosphate, 100 mM KCl and 1 mM EDTA (pH 7.6) and 5 μL of the 1.44 mMsecondary inhibitor stock solution (2× stock). The mixture was vortexedand incubated for 30 min (25° C.). After 30 min, the pH was lowered to4.4 with 500 μL of 200 mM acetate, 100 mM KCl, 1 mM EDTA (pH 4.2). Thefinal 1 mL solution was vortexed and incubated at 37° C. withoutagitation. The solutions were vortexed and turbidity at 350 and 400 nmwas measured. UV spectra were collected at the following intervals: 0,4, 8, 24, 48, 72, 96, 120, 144, 168 and 192 h after acidification. Acontrol sample containing 5 μL of DMSO was prepared for comparison, andthe spectra were collected at the time points above. Each inhibitorsolution was prepared in groups of 10 to prevent disturbance of thecuvettes before a reading was taken. After a UV absorbance was obtained,the cuvettes corresponding to that time-point were discarded. The fibrilformation assay employing equimolar (3.6 μM) TTR and inhibitorconcentration was performed as above using a 1× secondary inhibitorstock solution prepared as follows: A stock solution was prepared byten-fold dilution of the 7.2 mM 10× primary stock solution with DMSO toa final concentration of 0.72 mM and used in the fibril formation assayas described above. All compounds were found to be soluble throughoutthe course of the experiment, ensuring that turbidity was the result ofTTR amyloid formation.

The compounds described were evaluated as TTR amyloid fibril inhibitorsusing a turbidity assay. WT TTR amyloidosis was initiated byacidification of TTR preincubated with inhibitor (25° C., 30 min),employing buffer addition to jump the pH to a final value of 4.4. Afterincubation of each mixture for 72 h (37° C.), the turbidity was measuredat 350 and 400 nm using a UV-vis spectrometer. All amyloid fibrilformation data was normalized to WT TTR amyloidogenesis in the absenceof inhibitor, assigned to be 100% fibril formation. Therefore, 5% fibrilformation corresponds to a compound inhibiting 95% of WT TTR fibrilformation after 72 h. Each potential inhibitor was first evaluated at aconcentration of 7.2 μM relative to a TTR tetramer concentration of 3.6μM. Compounds allowing less than 15% fibril formation were reevaluatedat a concentration equal to the TTR concentration (3.6 μM) to select forthe inhibitors with the highest efficacy. Fibril formation of less than40% under these conditions is characteristic of a very good inhibitor,whereas 40-70% inhibition is indicative of a modest compound.

Inhibitors that keep TTR fibril formation below 50% at a concentrationequal to that of TTR (3.6 μM) were further evaluated for their abilityto bind TTR selectively over all other proteins in blood plasma. Thediflunisal concentration in blood can exceed 30 μM 20 h after a single500 mg dose, or 300 μM 4 h after the same dose. While this high level ofsustained plasma concentration suggests excellent bioavailability, moreselective inhibitors will allow for lower dosing and potentially fewerside-effects; therefore, human plasma was incubated with this subset ofinhibitors at a final concentration of 10.8 μM (average TTRconcentration in human plasma is approximately 5 μM). TTR was thencaptured using a resin-bound antibody, and the immobilized TTR waswashed three times with a solution of TSA (tris saline)/0.05% saponin,followed by two washes with TSA. The TTR-inhibitor complex was liberatedfrom the resin with 100 mM triethylamine (pH 11.5), and thestoichiometry of inhibitor present relative to TTR was determined byreverse-phase HPLC analysis. A maximum of 2 equiv of inhibitor may bebound per TTR tetramer.

Materials and Methods

Transthyretin Antibody Purification and Conjugation to Sepharose

Antibodies were produced, purified and coupled to Sepharose. The resinwas stored as a 1:1 slurry in TSA (10 mM Tris, pH 8.0/140 mM NaCl/0.025%NaN₃). In addition, quenched Sepharose was prepared by coupling 200 mMTris, pH 8.0 to the resin instead of the antibody.

Human Plasma Preparation

Whole blood was drawn from healthy volunteers at the Scripps GeneralClinical Research Center's Normal Blood-Drawing Program and transferredto 50 mL conical tubes. The tubes were centrifuged at 3000 RPM (1730×g)in a Sorvall RT7 benchtop centrifuge equipped with a swinging bucketrotor for 10 min at 25° C. The plasma supernatant was removed andcentrifuged again at 3000 RPM for 10 min to remove the remaining cells.Sodium azide was added to give a 0.05% solution. The plasma was storedat 4° C. until use

Immunoprecipitation of Transthyretin and Bound Compounds

A 2 mL eppendorf tube was filled with 1.5 mL of human blood plasma and7.5 μL of a 2.16 mM DMSO solution of the compound under evaluation. Thissolution was incubated at 37° C. for 24 h. A 1:1 resin/TSA slurry (187μL) of quenched Sepharose was added to the solution and gently rocked at4° C. for 1 h. The solution was centrifuged (16,000×g) and thesupernatant divided into 3 aliquots of 400 μL each. These were eachadded to 200 μL of a 1:1 resin/TSA slurry of the anti-transthyretinantibody-conjugated Sepharose and slowly rocked at 4° C. for 20 min. Thesamples were centrifuged (16,000×g) and the supernatant removed. Theresin was washed with 1 mL TSA/0.05% Saponin (Acros) (3×10 min) at 4°C., and additionally with 1 mL TSA (2×10 min) at 4° C. The samples werecentrifuged (16,000×g), the final wash removed, and 155 μL of 100 mMtriethylamine, pH 11.5 was added to elute the TTR and bound smallmolecules from the antibodies. Following gentle rocking at 4° C. for 30min, the samples were centrifuged (16,000×g) and 145 μL of thesupernatant, containing TTR and inhibitor, was removed.

HPLC Analysis and Quantification of Transthyretin and Bound Compounds

The supernatant elution samples from the TTR antibody beads (145 μL)were loaded onto a Waters 71P autosampler. A 135 μL injection of eachsample was separated on a Keystone 3 cm C18 reverse phase columnutilizing a 40-100% B gradient over 8 min (A: 94.8% H₂O/5%acetonitrile/0.2% TFA; B: 94.8% acetonitrile/5% H₂O/0.2% TFA),controlled by a Waters 600E multisolvent delivery system. Detection wasaccomplished at 280 nm with a Waters 486 tunable absorbance detector,and the peaks were integrated to give the area of both TTR and the smallmolecule. In order to determine the quantity of each species, knownamounts of tetrameric TTR or compound were injected onto the HPLC. Thepeaks were integrated to create calibration curves from linearregressions of the data using Kaleidagraph (Synergy Software). Thecalibration curves were used to determine the number of moles of eachspecies present in the plasma samples. The ratio of small molecule toprotein was calculated to yield the stoichiometry of small moleculebound to TTR in plasma.

Transthyretin Amyloid Fibril Formation Assay

The compounds were dissolved in DMSO at a concentration of 720 μM. FiveμL of a solution of the compound being evaluated was added to 0.5 mL ofa 7.2 μM TTR solution in 10 mM phosphate pH 7.6, 100 mM KCl, 1 mM EDTAbuffer, allowing the compound to incubate with TTR for 30 min. 495 μL of0.2 mM acetate pH 4.2, 100 mM KCl, 1 mM EDTA was added, to yield finalprotein and inhibitor concentrations of 3.6 μM each and a pH of 4.4. Themixture was then incubated at 37° C. for 72 h, after which the tubeswere vortexed for 3 sec and the optical density was measured at 400 nm.The extent of fibril formation was determined by normalizing eachoptical density by that of TTR without inhibitor, defined to be 100%fibril formation. Control solutions of each compound in the absence ofTTR were also tested and none absorbed appreciably at 400 nm.

Crystallization and X-ray Data Collection

Crystals of recombinant TTR were obtained from protein solutions at 5mg/ml (in 100 mM KCl, 100 mM phosphate, pH 7.4, 1 M ammonium sulfate)equilibrated against 2 M ammonium sulfate in hanging drop experiments.The TTR•ligand complexes were prepared from crystals soaked for 2 weekswith a 10-fold molar excess of the ligand to ensure full saturation ofboth binding sites. 1:1 acetone:water solution was used as a soakingagent. A DIP2030b imaging plate system (MAC Science, Yokohama, Japan)coupled to a RU200 rotating anode X-ray generator was used for datacollection. The crystals were placed in paratone oil as acryo-protectant and cooled to 120 K for the diffraction experiments.Crystals of all TTR•ligand complexes are isomorphous with the apocrystal form containing unit cell dimensions a=43 Å, b=86 Å and c=65 Å.They belong to the space group P2₁2₁2 and contain half of thehomotetramer in the asymmetric unit. Data were reduced with DENZO andSCALEPACK.

Structure Determination and Refinement

The protein atomic coordinates for TTR from the Protein Data Bank(accession number 1BMZ) were used as a starting model for the refinementof native TTR and the TTR-ligand complexes by molecular dynamics andenergy minimization using the program CNS. Maps were calculated fromdiffraction data collected on TTR crystals either soaked with compoundsor cocrystalized simultaneously. For the complexes of TTR with thecompounds, the resulting maps revealed approximate positions of theligand in both binding pockets of the TTR tetramer, with peak heights ofabove 5-9 r.m.s. In order to further improve the small molecule electrondensity and remove the model bias, the model was subjected to severalcycles of the warp/shake protocol, which resulted in noticeableimprovement in the map, especially around the inhibitor. Subsequentmodel fitting was done using these maps and the ligand molecule wasplaced into the density. In all three cases the minimum-energyconformation of the inhibitor calculated by the program InsightII(Accelrys) was in good agreement with the map. Because of the two-foldcrystallographic symmetry axis along the binding channel, a statisticaldisorder model must be applied, giving rise to two ligand binding modesin each of the two binding sites of tetrameric TTR. Water molecules wereadded based upon the unbiased electron density map. Because of the lackof interpretable electron densities in the final map, the nineN-terminal and three C-terminal residues were not included in the finalmodel.

Since modifications would be apparent to those of skill in the art, thesubject matter claimed herein is intended to be limited only by thescope of the appended claims.

1. A compound of formulae I:

or a pharmaceutically acceptable derivative thereof, wherein: Y is COOH,COOR⁵, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂, CONHSO₂Ar, CONHCH(R⁶)COOH,OH, CH₂OH or —(CH₂)_(n)—C(R⁶)(NH₂)—COOH; X¹ is O, S, S(O), S(O)₂,—O—N═CH— or NR¹¹; R¹, R² and R³ are each independently selected fromhydrogen, halo, OR⁵, OAr, OHet, OCH₂Ar, OCH₂Het, CN, B(OH)₂, CONR⁷R⁸,alkyl, —(CR⁹R¹⁰)_(n)OH, —(CR⁹R¹⁰)_(n)NR⁷R⁸, —(CR⁹R¹⁰)_(n)SH or CF₃; Hetis heteroaryl, optionally substituted with halo, OR, alkyl or haloalkyl;Ar is aryl, optionally substituted with halo, OR, alkyl or haloalkyl; Ris hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl orheteroaryl; R⁴ is hydrogen, halo, OH or alkyl; R⁵ is alkyl, haloalkyl,cycloalkyl or heterocyclyl; R⁶ is the side chain of a naturallyoccurring α-amino carboxylic acid; R⁷ and R⁸ are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl orheteroaryl; R⁹ and R¹⁰ are each independently hydrogen, halo, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; R¹¹ ishydrogen or alkyl; and n is an integer from 0-3; with the provisos that(i) when Y is COOH and is in the 3 or 4 position, then R¹, R² and R³ arenot Cl, CF₃ or F; (ii) when Y is COOH, then R¹, R² and R³ are not allhydrogen; (iii) when Y is COOH and is in the 2 position, then R¹, R² andR³ are not Cl, CH₃ or CF₃; (iv) when Y is OH and is in the 3 or 4position, then R¹, R² and R³ are not all hydrogen; (v) when Y is OH andis in the 3 or 4 position, then R¹, R² and R³ are not fluoro; (vi) whenY is CH₂OH, then R¹, R² and R³ are not all hydrogen; (vii) when Y isCH₂OH, then R¹, R² and R³ are not chloro.
 2. The compound of claim 1,wherein: Y is COOH, tetrazolyl, CONHOH, B(OH)₂ or OH; X¹ is O, —O—N═CH—or NH; R⁴ is hydrogen or OH; and R¹, R² and R³ are each independentlyselected from hydrogen, halo, OH, B(OH)₂ or CF₃.
 3. The compound ofclaim 1, wherein Y is COOH.
 4. The compound of claim 1, wherein Y istetrazolyl, B(OH)₂ or CONHOH.
 5. The compound of claim 1, wherein Y isOH.
 6. The compound of claim 1, wherein R¹, R² and R³ are eachindependently selected from hydrogen, halo and OH.
 7. The compound ofclaim 1, wherein R¹, R² and R³ are each independently selected fromhydrogen and CF₃.
 8. The compound of claim 1, wherein R¹ and R² are eachindependently hydrogen and R³ is CF₃.
 9. The compound of claim 1,wherein R¹, R² and R³ are each independently selected from hydrogen,halo and B(OH)₂.
 10. The compound of claim 1, wherein R¹, R² and R³ areeach independently selected from hydrogen, Br and I.
 11. The compound ofclaim 1, wherein R¹, R² and R³ are each independently selected from haloand OH.
 12. The compound of claim 1, wherein R¹, R² and R³ are eachindependently selected from halo and B(OH)₂.
 13. The compound of claim1, wherein R¹ and R² are each halo and R³ is OH.
 14. The compound ofclaim 1, wherein R¹ and R² are each halo and R³ is B(OH)₂.
 15. Thecompound of claim 1, wherein R¹ and R² are each halo and R³ is H. 16.The compound of claim 1, wherein Het is pyrimidine, pyridine orthiophene.
 17. The compound of claim 1, wherein X¹ is O or NH.
 18. Thecompound of claim 1, wherein X¹ is —O—N═CH—.
 19. The compound of claim1, wherein R⁴ is hydrogen or OH.
 20. The compound of claim 1, wherein R⁴is hydrogen.
 21. The compound of claim 1, wherein R⁴ is OH.
 22. Thecompound of claim 1 that has the formulae:


23. A pharmaceutical composition, comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 24. The pharmaceutical compositionof claim 23 formulated for single dosage administration.
 25. A methodfor the stabilization of transthyretin in a tissue or in a biologicalfluid, comprising administration of a compound of claim
 1. 26. A methodof inhibiting transthyretin misfolding, comprising contacting thetransthyretin with a compound of claim
 1. 27. A method of treating,preventing, or ameliorating one or more symptoms of a transthyretinamyloid disease, comprising administering a compound of claim
 1. 28. Amethod of preventing dissociation of a transthyretin tetramer by kineticstabilization of the native state of the transthyretin tetramer,comprising contacting the tetramer with a compound of claim
 1. 29. Themethod of claim 27, wherien the transthyretin amyloid disease isfamilial amyloid polyneuropathy, familial amyloid cardiomyopathy, orsenile systemic amyloidosis.
 30. The method of claim 27, wherein thedisease is Alzheimer's disease, spongiform encephalopathy,polyneuropathy, type II diabetes and medullary carcinoma of the thyroid.31. A method of treating, preventing, or ameliorating one or moresymptoms of a transthyretin mediated disease or disorder, comprisingadministering a compound of claim
 1. 32. The method of claim 31, whereinthe disease is obesity.
 33. A method of stabilizing a transthyretintetramer, comprising contacting the tetramer with a compound of claim 1.34. A method of inhibiting formation of TTR amyloid, comprisingadministering a compound of claim 1.